The conventional PCR technique quantifies the amplicon (product of amplification) at the end of the process. By only measuring the amplicon at the end of PCR (endpoint detection), the relationship between the starting amplicon and the end amplicon becomes inaccurate. This is caused by a multitude of factors including the presence of inhibitors, reagent limitation, and the presence of pyrophosphate molecules. Real-time PCR (RT-PCR) is a more reliable way to quantify the relationship between the starting amount of amplicon and the amplicon produced by the PCR process. In RT-PCR, the quantity of the amplicon is monitored throughout the entire process, which can show the relationship between the starting amount of target sequences and the amount after amplification (Artika et al.). By using RT-PCR, you can quantify how much of a target virus, or bacteria is present in a sample by using reference standards, which is why RT-PCR is also called quantitative PCR (qPCR) (Salipante et al.).
In order to monitor the quantity of a target sequence in RT-PCR, fluorescent probes are utilized. There are two different types of these probes: SYBR Green and EvaGreen probes, and TaqMan probes. SYBR Green probes settle in between the bases of the DNA helix which allow non-specific and specific amplicons to be monitored. TaqMan probes link onto specific oligonucleotides instead of base pairs which allow for high specificity.
One major application of RT-PCR is its ability to be used as a confirmatory test for a virus or bacteria. (Akingbola et al., Kralick et al.). This was utilized to identify COVID-19 for the first time in Wuhan, China. In order for a test like this to be positive, the cycle threshold value or the number of cycles it takes to detect a virus, should be <40. A CT value of <40 indicates a high viral load or a positive indicator of the virus (Artika et al.). This method has also been utilized when diagnosing the bacteria Vibrio cholerae, which causes Cholera (Akingbola et al.). Using RT-PCR to test water is valuable when monitoring the environment (Akingbola et al., Tiwari et al.) because using CT values can uncover Chlorea in bodies of water, which can allow for early response to reduce the effects of the disease (Akingbola et al.).